2.2 Site-specific PEGylation of FGF2 conjugates and purification
The PEGylated FGF2 conjugates were generated by site specific PEGylation using mPEG-Maleimide (mPEG-MAL, 5kDa). We optimized the reaction conditions, including temperature, reaction time, and the PEG-to-FGF2 molar ratio as 4 °C a PEG-to-FGF2 molar ratio of 4, and 1 h, respectively. The yield of PEGylation was confirmed by SDS-PAGE.
To separate the PEGylated FGF2 conjugates from unreacted FGF2 and mPEG-MAL, reaction mixtures were applied at a rate of 5 mL/min to a Heparin HP column (5 mL bed volume) pre-equilibrated with 15 column volumes (CVs) of binding buffer (25 mM HEPES buffer, pH 7.5). The column was then washed with 10 CVs of binding buffer, and then the protein was eluted with buffer A (25 mM HEPES buffer, pH 7.5 and buffer B (25 mM HEPES buffer, pH 7.5, 2 M NaCl) over 30 CVs. Eluted fractions were collected and analyzed by SDS-PAGE.